Endogenous fragment of hemoglobin, neokyotorphin, as cell growth factor.

It is shown that neokyotorphin (the alpha-globin fragment 137-141) stimulates proliferation of normal cells (murine embryonic fibroblasts, red bone marrow and spleen cells) and tumor cells (murine melanoma and transformed fibroblasts L929) in the absence or in the presence of fetal bovine serum. In contrast to serum deprivation conditions, the ability to potentiate L929 cell growth in the presence of fetal serum is strongly cell density dependent. The peptide also enhances the viability of L929 cells, murine embryonic fibroblasts and of the primary cultures of murine red bone marrow cells and splenocytes under serum-deprivation conditions for at least 72 h. The results of flow cytometry analysis suggest that the effect of neokyotorphin on survival of L929 cells in serum-free culture medium is due to maintenance of cell proliferation in the absence of growth factors. Along with cell cycle progression the peptide induces reversible reduction of L929 cell size.

Peptides comprising the bulk of rat brain extracts: isolation, amino acid sequences and biological activity.

Chromatographic separation of rat brain extracts followed by automatic Edman sequencing of the major individual components resulted in identification of 61 endogenous peptides derived from known functional proteins (hemoglobin, myelin basic protein, cytochrome-c oxidase, etc.) or unknown precursors. The results are compared with the data obtained earlier for bovine brain. Although the sequences of bovine and rat hemoglobin contain about 20% of amino acid substitutions, the families of structurally related peptides are very similar in both extracts. Several other proteins also give rise to identical or closely related peptide fragments in the two mammalian species. The outlined similarity extends almost exclusively to the most abundant peptides present in the extracts. The minor components show less overlap. Four hemoglobin-derived peptides isolated from rat brain were shown to be biologically active in tumor cells. Eleven are identical to bioactive peptides from other species. Ten structurally overlap with bioactive peptides from other sources. The data obtained show similar biosynthetic pathways of pool components in different species, the resultant peptides being aimed at fulfilling related functions.

GABA-induced changes of the tissue-specific peptide pool of white rat brain.

Internasal administration of gamma-aminobutyric acid (GABA) induced prolonged behaviour changes and the appearance of three new compounds absent in the brain extracts of control rats. Two peptides associated with GABA administration were isolated and sequenced: Thr-Tyr-Thr-Phe, which corresponds to a gamma-immunoglobulin segment, and Val-Leu, which is present in a great number of proteins, hence its precursor could not be established. The third compound was not amenable to the Edman degradation technique. The data obtained show that the introduction of a neurotransmitter could cause specific changes in the levels of tissue-specific peptide components.

LVV- and VV-hemorphins: comparative levels in rat tissues.

Screening of hemorphins in extracts of rat lung, brain, heart and spleen was carried out. The threshold for detection of hemorphins was 0.01 nmol for spleen and 0.05 nmol for other tissues. Both the content and the composition of hemorphins differed significantly in the tissues analyzed. Heart and lung extracts were rich in these peptides, the content of the most abundant components reaching 16-44 nmol/g of tissue. In contrast, spleen and brain contained much lower amounts of hemorphins, i.e. about 0.3-2.6 nmol/g of tissue. The most represented hemorphin in lung, heart and brain was VV-hemorphin-5, while the content of other members of the hemorphin family depended significantly on the tissue analyzed: lung extract was also rich in LVV-hemorphin-5, heart contained similar amounts of LVV-hemorphin-7 and LVV-hemorphin-5 and brain of LVV-hemorphin-6. In contrast, the hemorphin family in spleen was represented mainly by C-terminally shortened VV-hemorphins, i.e. VV-hemorphin-4 and VV-hemorphin-3. The levels of hemorphins in all cases were sufficient to activate the opioid receptors of the respective tissues.

Peptides from bovine brain: structure and biological role.

Fractionation of bovine brain extracts followed by automatic Edman sequencing of individual components resulted in identification of 107 endogenous peptides formed from functional proteins (haemoglobin, myelin basic protein, cytochrome c oxidase, etc) or unknown precursors. Several of the newly identified brain peptides demonstrate different types of biological activity; some of the substances show considerable overlap with the known biologically active peptides. It is suggested that these peptides should participate in regulation of extracellular and intracellular biochemical processes. A concept of 'tissue-specific peptide pool' is formulated describing a novel system of peptidergic regulation, complementary to the conventional hormonal and neuromodulatory systems. According to that description functional proteins provide their proteolytically derived fragments for maintaining the tissue homeostasis by modulating the availability of peptide receptors to respective 'true' ligands.

Neokyotorphin and neokyotorphin (1-4): secretion by erythrocytes and regulation of tumor cell growth.

Human erythrocytes release neokyotorphin, the 137-141 fragment of hemoglobin alpha-chain into the supernatant of red blood cells primary culture. However, the neokyotorphin fragment 1-4 that is formed together with neokyotorphin inside the red blood cells and in various tissues is not found in the supernatant. Both neokyotorphin and its 1-4 fragment were shown to stimulate proliferation of L929 tumor cells.

Hemoglobin as a source of endogenous bioactive peptides: the concept of tissue-specific peptide pool.

Scattered literature data on biologically active hemoglobin-derived peptides are collected in the form of tables. Respective structure-functional correlations are analyzed and the general conclusion is reached that hemoglobin fragments must have a profound physiological function. Evidence is presented that generation of hemoglobin fragments starts inside the erythrocytes. At that stage alpha- and beta-globin chains of hemoglobin predominantly give rise to relatively long peptides containing ca. 30 amino acid residues. The primary proteolysis is followed by the next degradation step coupled with excretion of newly formed shorter peptides form red blood cells. Both the primary and the secondary proteolysis products are subjected to further stepwise C- and N-terminal chain shortening, giving rise to families of closely related peptides that are actually found in animal tissue extracts. The possible sites of primary proteolysis are compared with the positions of the exposed secondary structure elements within the monomeric alpha- and beta-globins as well as the tetrameric hemoglobin. Two tentative schemes are proposed for hemoglobin degradation, one of which starts at the globin loops exposed on the surface of the tetramer and the other, at monomeric globins where more sites are available for the action of proteases. The concept of a "tissue-specific peptide pool" is formulated, describing a novel system of peptidergic regulation, complementary to the conventional hormonal and neuromodulatory systems. According to that description, hemoglobin is only a single example, although an important one, of a vast number of functional proteins providing their proteolytically derived fragments for maintaining the tissue homeostasis.

Alteration of intraerythrocyte proteolytic degradation of hemoglobin during Hodgkin's disease.

The erythrocyte lysate samples obtained from 10 healthy donors (aged of 23 +/- 12 years) and 16 patients with Hodgkin's disease (aged of 39 +/- 25 years) with the following histological types: 12 mixed cellularity and 4 nodular sclerosis, were studied. Patients with Hodgkin's disease (HD) were randomly selected before, during or after the completion of combined chemotherapy. A comparative analysis of peptide components of erythrocyte lysate samples of HD patients and healthy donors was carried out. The amino acid sequences of 4 peptides, corresponding to fragments 1-33, 1-32, 1-31 and 1-30 of human hemoglobin (Hb) alpha-chain were determined. Increase of the content of two fragments corresponding to 1-31 and 1-32 amino acid residues of alpha-globin were detected for HD patients. The link between the normal process of proteolytic degradation and those occurring during HD is proposed. The possibility of using the identified alterations recorded during HD diagnosis is discussed.

Neokyotorphin and neokyotorphin (1-4): cytolytic activity and comparative levels in rat tissues.

The content of biologically active hemoglobin fragment neokyotorphin (TSKYR) as well as that of neokyotorphin fragment (1-4) (TSKY) were determined in extracts of lung, heart, and brain tissue of rats. The content of both peptides as well as the neokyotorphin/neokyotorphin(1-4) ratio differed significantly from each other in these tissues. The respective parameters deviate considerably from those of erythocytes where these peptides are originally formed. Comparative analysis of cytolytic activity of peptides was performed at human erythroid leukaemia (K562) and murine transformed fibroblast (L929) cell lines. TSKY showed reliable cytolytic activity in both cell lines, while neokytorphin was not cytotoxic. The data obtained lead to speculation that endogenous hemoglobin fragments might participate in regulation of tumor growth in vivo.

Proteolytic degradation of hemoglobin in erythrocytes leads to biologically active peptides.

A number of hemoglobin-derived homogeneous peptides were isolated from erythrocyte lysate. The amino acid sequences of nine peptides were determined. Seven out of nine peptides were relatively long, 30-32 membered peptides covering the N- or C-terminal sequences of globin chains. The remaining two were the pentapeptide neo-kyotorphin and its tetrapeptide derivative.

Isolation of endogenous hemorphin-related hemoglobin fragments from bovine brain.

Six short-chain peptides were isolated from an acidic extract of bovine brain in the course of total peptide screening. Their primary structures determined by Edman degradation were LVVYP, LVVYPWT, LVVYPWTQ, LVVYPWTQRF, VVYPWTQ and VVYPWTQRF, which respectively corresponded to the fragments 31-35, 31-37,31- 38, 31-40, 32-38 and 32-40 of bovine hemoglobin beta-chain. All these peptides contained sequences of opioid peptides - hemorphins. For two of these peptides, viz. 32-38 and 31-40, isolated from other sources, an opioid activity was demonstrated formerly.